e u r o p e a n j o u r n a l o f p a e d i a t r i c n e u r o l o g y x x x ( 2 0 1 3 ) 1 e1 1

Official Journal of the European Paediatric Neurology Society

Original article

Confirmation of chromosomal microarray as a first-tier

clinical diagnostic test for individuals with developmental
delay, intellectual disability, autism spectrum disorders and
dysmorphic features5,55

Agatino Battaglia a,*, Viola Doccini a, Laura Bernardini b, Antonio Novelli b, Sara Loddo b,
Anna Capalbo b, Tiziana Filippi a, John C. Carey c
a
Stella Maris Clinical Research Institute for Child and Adolescent Neuropsychiatry, via dei Giacinti, 2, 56128 Calambrone, Pisa, Italy
b
Mendel Laboratory, IRCCS Casa Sollievo della Sofferenza Hospital, San Giovanni Rotondo (FG), Italy
c
Division of Medical Genetics, Dept. of Pediatrics, University of Utah Health Sciences Center, Salt Lake City, UT, USA

article info abstract

Article history: Background and objectives: Submicroscopic chromosomal rearrangements are the most
Received 30 October 2012 common identifiable causes of intellectual disability and autism spectrum disorders
Received in revised form associated with dysmorphic features. Chromosomal microarray (CMA) can detect copy
28 February 2013 number variants <1 Mb and identifies size and presence of known genes. The aim of this
Accepted 28 April 2013 study was to demonstrate the usefulness of CMA, as a first-tier tool in detecting the eti-
ology of unexplained intellectual disability/autism spectrum disorders (ID/ASDs) associ-
Keywords: ated with dysmorphic features in a large cohort of pediatric patients.
Chromosomal microarray (CMA) Patients and methods: We studied 349 individuals; 223 males, 126 females, aged 5 months-19
Array-CGH years. Blood samples were analyzed with CMA at a resolution ranging from 1 Mb to 40 Kb.
Developmental delay The imbalance was confirmed by FISH or qPCR. We considered copy number variants (CNVs)
Intellectual disability causative if the variant was responsible for a known syndrome, encompassed gene/s of
Neurodevelopmental disorders known function, occurred de novo or, if inherited, the parent was variably affected, and/or the
Autism spectrum disorders involved gene/s had been reported in association with ID/ASDs in dedicated databases.
Dysmorphic features Results: 91 CNVs were detected in 77 (22.06%) patients: 5 (6.49%) of those presenting with
borderline cognitive impairment, 54 (70.13%) with a variable degree of DD/ID, and 18/77
(23.38%) with ID of variable degree and ASDs. 16/77 (20.8%) patients had two different
rearrangements. Deletions exceeded duplications (58 versus 33); 45.05% (41/91) of the
detected CNVs were de novo, 45.05% (41/91) inherited, and 9.9% (9/91) unknown. The CNVs

Abbreviations: ASDs, autism spectrum disorders; CMA, chromosomal microarray; DD, developmental delay; ID, intellectual disability.
5
Whats known on this subject: Recommendations in medical genetics practice call for the use of chromosomal microarray as a first-
tier clinical diagnostic test for individuals with developmental disabilities or congenital anomalies. The recommendations were based
on reports dealing with heterogeneous populations studied with a variety of protocols.
55
What this study adds: After comprehensive phenotypic characterization at one tertiary center including a standardized psycho-
pathological assessment for Developmental Delay/Intellectual Disability/Autism Spectrum Disorders, we detected a high diagnostic
yield of Chromosomal Microarray in children with unexplained Intellectual Disability/Autism Spectrum Disorders/dysmorphic features,
confirming its value as first-tier tool in the pediatric setting.
* Corresponding author. Tel.: 39 (0)50 886248.
E-mail address: agatino.battaglia@inpe.unipi.it (A. Battaglia).
1090-3798/$ e see front matter 2013 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.ejpn.2013.04.010

Please cite this article in press as: Battaglia A, et al., Confirmation of chromosomal microarray as a first-tier clinical diagnostic
test for individuals with developmental delay, intellectual disability, autism spectrum disorders and dysmorphic features,
European Journal of Paediatric Neurology (2013), http://dx.doi.org/10.1016/j.ejpn.2013.04.010
2 e u r o p e a n j o u r n a l o f p a e d i a t r i c n e u r o l o g y x x x ( 2 0 1 3 ) 1 e1 1

caused the phenotype in 57/77 (74%) patients; 12/57 (21.05%) had ASDs/ID, and 45/57
(78.95%) had DD/ID.
Conclusions: Our study provides further evidence of the high diagnostic yield of CMA for
genetic testing in children with unexplained ID/ASDs who had dysmorphic features. We
confirm the value of CMA as the first-tier tool in the assessment of those conditions in the
pediatric setting.
2013 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights
reserved.

1. Introduction genetic technology, and their application. Since microarray-

Clinical genetic testing, including high-resolution array
comparative genomic hybridization (array CGH) or chromo-
somal microarray (CMA), has been suggested as a standard
practice for children with diagnoses including unexplained
developmental delay/intellectual disability (DD/ID), autism
spectrum disorders (ASDs), and dysmorphic features. The
incidence of DD/ID in the general population is estimated to be
3%.1,2 It is a broad diagnosis encompassing a wide variety of
phenotypes and severity. Clinically, DD/ID is characterized by
a significant impairment of cognitive and adaptive functions,
with onset before age 18 years.3e5 ID may become evident
during infancy or early childhood as developmental delay
(DD), but it is best diagnosed during the school years. Ac-
cording to the intelligence quotient (IQ), obtained by assess-
ment with one or more of the standardized individually
administered tests, ID is sub-grouped in five degrees of
severity: borderline ID (IQ level 70e85); mild ID (IQ level 50e55
to approximately 70); moderate ID (IQ level 35e40 to 50e55);
severe ID (IQ level 20e25 to 35e40) and profound ID (IQ level
below 20e25). Usually the presenting symptoms are impair-
ments in adaptive functioning.6 ASDs are a clinically hetero-
geneous group of disorders including autism, Asperger
syndrome, pervasive developmental disorders not otherwise
specified (PDD-NOS), and childhood disintegrative disorder.7
They are characterized by markedly abnormal or impaired
development in social interaction, and communication, and a
markedly restricted repertoire of activity and interests, and
can co-occur with ID. ID/ASDs are a condition of great concern
for public health and society and represent a limitation, of
variable degree, in all fields of daily living for the affected in-
dividual and his/her family. The cost of care over a lifetime
can be high.8 Beyond the financial burden, caring for a child/
adolescent with DD/ID/ASDs exerts substantial social and
emotional effects on the other family members. Knowing the
genetic cause of DD/ID/ASDs in a child is of importance to the
parents; as a diagnosis can bring comfort to them; allows for
prediction of the clinical evolution with relative certainty, and
informs an appropriate rehabilitation plan and maintenance
of general health. Additionally a diagnosis usually helps in
establishing an accurate recurrence risk.6 Most individuals
lack enough specific history or features from physical exam-
ination to suggest a definite genetic or non-genetic etiology.
Before the CMA era, there was a marked variation in reported
etiologic yield of DD/ID (from 10% to 80%)9e16 reflecting vari-
ations in sample population characteristics, methods of clas-
sification and diagnosis, the availability of imaging and
based genomic copy-number analysis has been used as a
clinical genetic test for this patient population, the reported
variation in etiologic yield has been 5%e35%.17 Understanding
the genetics of autism has proven difficult as well. When
considered as a single entity, ASDs does not fit the usual in-
heritance patterns. Recently, a consensus statement recom-
mended CMA as the first-tier clinical diagnostic test for
individuals with DD/ID/ASDs or congenital anomalies.17 CMA
has dramatically changed the nature of human genome
analysis as it detects rearrangements as small as 1 Mb or less,
and identifies size and presence of known genes, and helps to
characterize small marker chromosomes.18 The aim of this
study is to confirm the usefulness of CMA as a first-tier clinical
diagnostic test in the evaluation of children with apparently
idiopathic DD/ID/ASDs and dysmorphic features, in whom
standard karyotype/FISH and, when appropriate, molecular
analysis at the FRAXA/E loci, were normal.
2. Subjects and methods
The study was approved by the institutional review board of
the Stella Maris Institute, and appropriate informed consent
was obtained from all parents/guardians of our patients.
We recruited 349 patients affected by DD/ID/ASDs/dysmor-
phic features of unknown origin observed at the Stella Maris
Institute between May 2004 and December 2011. All, but one, had
already received standard cytogenetics or FISH and, when
appropriate, molecular analysis at the FRAXA/E loci. All patients
underwent a consistent and rigorous study protocol including an
accurate prenatal/birth history and family history, together with a
three-generation pedigree, brain MRI, EEG, metabolic work-up,
complete cognitive evaluation, a thorough physical and neuro-
logical examination (AB and JCC.), and, when appropriate, a psy-
chiatric assessment. The children referred with a provisional
diagnosis of ASD were also evaluated with the ADI-R19 and ADOS-
G.20 In all patients, a chromosomal aberration was suspected
because all assessed individuals showed signs of a dysmorphic
syndrome: three or more minor anomalies; two major malfor-
mations; or facial features different from the family background.
There were 223 (63.9%) males and 126 (36.1%) females, aged
5 months to 19 years. Blood samples, collected from patients
and their parents, were sent to the Cytogenetic Laboratory of
the IRCCS Hospital S. Giovanni Rotondo/CSS Mendel Institute,
Rome, and were analyzed with CMA at a resolution ranging
from 1 Mb to 40 Kb. BAC-array (Integrachip 0.8 Mb, Tech-
nogenetics, Milan, Italy), oligonucleotide-array (Agilent 44 K or

Please cite this article in press as: Battaglia A, et al., Confirmation of chromosomal microarray as a first-tier clinical diagnostic
test for individuals with developmental delay, intellectual disability, autism spectrum disorders and dysmorphic features,
European Journal of Paediatric Neurology (2013), http://dx.doi.org/10.1016/j.ejpn.2013.04.010
 e u r o p e a n j o u r n a l o f p a e d i a t r i c n e u r o l o g y x x x ( 2 0 1 3 ) 1 e1 1 3

180 K; Agilent technologies, Waldbronn, Germany) and/or SNP-
array platform (6.0 Chip; Affymetrix, Palo Alto, CA) were used
for CMA analysis; manufacturers suggestions were used for
experiments. Sex-matched pooled DNAs (Promega, Madison,
WI) were used as reference in BAC- and oligonucleotide-array
analysis, while the 270 International HapMap Project control
samples were used as a reference model in SNP-array
experiments.
All copy number changes were confirmed by FISH or
quantitative PCR. FISH analysis was carried out using com-
mercial probes (Vysis Inc., Downers Grove, IL) or BACs clones
selected from a genomic library (32 K library; BACPAC Re-
sources, Oakland, CA). DNA was extracted by Quantum Prep
MiniPrep Kit (BioRad, Hercules, CA) and SpectrumGreen-dUTP
or SpectrumOrange-dUTP labeled using the Nick Translation
kit (Vysis Inc.), according to the manufacturers protocol.
Metaphase spreads were obtained following standard pro-
cedures and FISH analysis was performed as described in
Bernardini et al., [2007].21 Quantitative-PCR (qPCR) was per-
formed using an ABI 7000 Sequence Detection System
(Applied Biosystem, Foster City, CA) and DNA-binding dye
SYBR Green (Invitrogen Corporation, Carlsbad, CA) as
described by Carbone et al., [2008].22 Primers were designed by
Primer Express 2.0 software (Applied Biosystems) and an
amplified fragment of the TERT locus was used as a reference.
After the confirmation test and the inheritance assess-
ment, CNVs were classified as pathogenic, benign or with an
unknown clinical significance based on the guidelines
suggested by Miller et al., [2010]17 and successively by the
American College of Medical Genetics.23
All samples analyzed with SNP-array and negative to CNVs
analysis were evaluated for LSH (Long Stretches of Homozy-
gosity) scattered along the genome. This analysis can even-
tually detect a rate of homozygosity higher than expected as a
marker of ancestral homozygosity or parental consanguinity,
with an increased risk for recessive diseases, or uniparental
disomy in relevant imprinted regions. The LSH analysis was
performed as suggested by Kearney and colleagues [2011],24
with minor modifications. The rate of homozygosity was
calculated as the sum of autosomal LSH >1 Mb in relation with
the autosomal genome covered by SNPs. In particular, Gen-
eChip 6.0 platform covers about 2,781,532 kbp (hg19 release).
3. Results
We identified 91 CNVs in 77/349 (22.06%) patients; 53/223
(23.76%) males and 24/126 (19.04%) females. Five of 77 (6.49%)
patients showed a borderline cognitive impairment, 54/77
(70.13%) variable degrees of DD/ID, and. 18 of 77 (23.38%) pa-
tients had an ASD associated with ID of variable degree (Table
1). A causative CNV was found in 57/77 (74.02%) children, 45
(79%) of whom presenting only ID of variable degree, and 12
(21%) also ASDs (Table 2). Sixteen of 77 (20.78%) patients had
two chromosome rearrangements (3 of whom with ASDs/ID),
in 10 of them on different chromosomes (Table 3aec).

Table 1 e Flow diagram showing the number of patients studied and the number and characteristics of the detected CNVs.

STUDY SAMPLE: 349; 223 (63.9%) Males, 126 (36.1%) Females

BORDERLINE ID: 34 (9.8%); MIXED ID: 237 (67.9%); ID + ASDs: 78 (22.3%);

21 (61.8%) Males, 13 (38.2%) Females 142 (59.9%) Males, 95 (40.1%) Females 60 (76.9%) Males, 18 (23.1%) Females

CNVs identified in 77 patients (22.06%)

BORDERLINE ID: 5 (6.49%) MIXED ID: 54 (70.13%) ID + ASDs: 18 (23.38%)

Males: 4/21 Female: 1/13 Males: 34/142 Females: 20/95 Males: 15/60 Females: 3/18
(19%) (7,69%) (23.94%) (21.05%) (25%) (16.67%)

CNVs: 7 CNVs: 64 CNVs: 20

CAUSATIVE CNVs: 3 (42.86%) CAUSATIVE CNVs: 51 (79.69%) CAUSATIVE CNVs: 11 (55%)

INHERITED CNVs: DE NOVO CNVs: INHERITED DE NOVO CNVs: INHERITED DE NOVO

0 (0.0%) 2 (66.7%) CNVs: 11 (21.6%) 36 (70.6%) CNVs: 6 (54.5%) CNVs. 3 (27.3%)

In 1 (33.3%) CNV the origin was unknown In 4 (7.8%) CNVs the origin was unknown In 2 (18.2%) CNVs the origin was unknown

Please cite this article in press as: Battaglia A, et al., Confirmation of chromosomal microarray as a first-tier clinical diagnostic
test for individuals with developmental delay, intellectual disability, autism spectrum disorders and dysmorphic features,
European Journal of Paediatric Neurology (2013), http://dx.doi.org/10.1016/j.ejpn.2013.04.010
4 e u r o p e a n j o u r n a l o f p a e d i a t r i c n e u r o l o g y x x x ( 2 0 1 3 ) 1 e1 1
Table 2 e Percentage of borderline ID, mixed ID, and ID/ASDs patients in whom different CNVs categories were found.
CNVs causative CNVs probably
of the phenotipe causative of the
phenotipe
Borderline ID: 5 patients 2/5 (40%) 1/5 (20%)
Mixed ID: 54 patients 43/54 (79.63%) 2/54 (3.70%)
ID and ASDs: 18 patients 12/18 (66.67%) 3/18 (16.66%)
In 8/77 (10.38%) patients the rearrangement involved a sex
chromosome. In 5/77 (6.49%) patients with an abnormal
standard karyotype/FISH or FRAXA-E, CMA was pursued
because the clinical phenotype did not correlate well with the
reported chromosome aberration (47,XYY; del 1p36; del 1q43-
qter; FRAXA premutation allele, Robertsonian 13; 14 trans-
location at amniocentesis) (Table 3aec).
Of the 91 detected CNVs, 65/91 (71.4%) were classified as
causative; 13/91 (14.3%) non causative; 5/91 (5.5%) uncertain;
and 8/91 (8.8%) probably causative (Tables 1 and 4). Fifty-eight
were deletions and 33 duplications.
The percentage for the different CNVs categories found in
DD/ID patients versus DD/ID/ASDs ones are reported in Table 4.
Forty five and zero five percent (41/91) of the identified
CNVs were inherited; 45.05% (41/91) de novo; while in 9.9% (9/
91) the origin was unknown. Twenty (48.8%) of the inherited
CNVs were deletions, and 21 (51.2%) duplications. Thirty-two
(78.1%) of the de novo CNVs were deletions, and 9 (21.9%) du-
plications. Seventeen of forty-one (41.5%) inherited CNVs
were causative (Table 1).
Thirty-seven of 91 CNVs (40.6%) were <1 Mb in size, 28/91
(30.7%) were 1e5 Mb, 12/91 (13.2%) were 5e10 Mb and 14/91
(15.5%) were >10 Mb. 7/37 CNVs < 1 Mb in size were de novo; 10
of the 14 CNVs > 10 Mb in size were de novo.
The largest CNV identified in our study sized 21 Mb, while
the smallest was 92 Kb. Most CNVs were detected by CMA at
250 Kb resolution.
The evaluation of LSH in the 20 patients evaluated by SNP-
array and negative to CNVs analysis disclosed in one sample a
rate of homozygosity of 25%, indicating a first-degree parental
relationship. All other patients displayed an average rate of
2.6%, consistent with the expected total length of LSH of the
general population.25
4. Discussion
We identified chromosomal rearrangements in 22.06% of our
patients; this figure is in keeping with literature data.17
Although the study sample was represented by more males
than females, the percentage with causative CNVs was almost
the same in the two sex groups, suggesting that other sex-
linked factors, could contribute to the higher male frequency
amongst DD/ID/ASDs individuals.
The 71.4% (65/91) (Table 4) of aberrations was considered
causative to the phenotype, because in each situation the CNV
was de novo, responsible for a known syndrome, expanded or
altered a CNV inherited from a parent, was identical to a CNV
inherited from an affected parent, was similar to a CNV in an
affected relative, was a CNV overlapping a genomic imbalance
Please cite this article in press as: Battaglia A, et al., Confirmation of chromosomal microarray as a first-tier clinical diagnostic
test for individuals with developmental delay, intellectual disability, autism spectrum disorders and dysmorphic features,
European Journal of Paediatric Neurology (2013), http://dx.doi.org/10.1016/j.ejpn.2013.04.010
CNVs of uncertain CNVs non-causative of the
significance phenotipe
0/5 (0.0%) 2/5 (40%)
3/54 (5.56%) 6/54 (11.11%)
2/18 (11.11%) 1/18 (5.56%)
defined by a high-resolution technology in a CNV database for
patients with DD/ID/ASDs/dysmorphic features, contained
morbid OMIM genes, was gene rich, was a deletion, was a
homozygous deletion (Table 3aec). The 8.8% (8/91) (Table 4)
was considered probably causative because the CNVs,
although paternally or maternally inherited, encompassed
genes, such as CNTNAP2 (involved in synapse formation and
maintenance),26 SOS2 (possible RAS activator)27,28 in patient 45
with a phenotype reminiscent of a Noonan-CFC syndrome, or
had already been associated with ID. The 5.5% (5/91) (Table 4)
was considered uncertain because the CNVs, although
parentally segregated and being either duplications (pat. 78;
Table 3), or deletions (pat. 32, 33; Table 3), encompassed genes
involved in synapse and dendritic spine formation, such as
NLGN4X (associated with ASDs),29 and IL1RAPL2 (located,
together with IL1RAPL1, to a region on the X chromosome,
associated with X-linked non-syndromic MR)30 (Table 3aec).
The 14.3% (13/91) (Table 4) was considered non-causative
because the CNV was identical to that inherited from a
healthy parent, was completely contained within genomic
imbalance defined by a high-resolution technology in a CNV
database of healthy individuals, was gene poor, was a dupli-
cation containing not known dosage-sensitive genes, was
devoid of known regulatory elements.17,23 The data related to
parentally segregated CNVs should be interpreted with
caution, because such imbalances could also contribute to the
probands phenotype through variable penetrance or expres-
sivity, or both; through epigenetic effects, or by uncovering a
recessive mutation on the non-deleted allele.
As previously reported,31 we observed a greater number of
deletions than duplications (58 versus 33). This can have a
dual explanation, both technical and biological. Technically,
there is a greater chance that some duplications may be
missed. Biologically, duplications generally cause a milder
phenotype, possibly leading to a selection bias. In addition,
the frequency of random duplications in the human genome
may be lower then that of deletions.32
Sixteen patients had two different chromosome rear-
rangements, in 10 of whom on different chromosomes (Table
3aec). This could explain the complexity of their clinical pic-
ture, and is in keeping with the two-hit model, in which one
CNV predisposes to neuropsychiatric phenotypes as a single
event and exacerbates neuro-developmental phenotypes in
association with the second one.33
In some cases the identified anomalies involved known genes
(Table 3aec), some of these related to well defined neuropsychi-
atric disorders such as RAI1 (SmitheMagenis syndrome), NF1
(neurofibromatosis 1), EDNRB (Waardenburg syndrome).
Although the diagnostic yield among the patients with
severe-profound ID would be expected to be higher than
 e u r o p e a n j o u r n a l o f p a e d i a t r i c n e u r o l o g y x x x ( 2 0 1 3 ) 1 e1 1 5

Table 3 e a, b, c. Summary of the clinical and molecular cytogenetic data of 77 individuals with CNVs.
Case Sex Age at Previously normal Previously Severity of Epilepsy Autism Dysmorphic
diagnosis karyotype/fish normal DD/ID features
fraxa/E

1 Female 3 yrs 9 ms Yes/FISH N.D. Moderate-severe No No Yes

subtel 1pVysis
2 Male 4 yrs Yes Yes Moderate-Mild No No Yes
3 Maleb 4 yrs 5 ms Yes Yes Mild No No Yes
4 Maleb 4 yrs 5 ms Yes Yes Borderline No PDD-NOS Yes
5 Female 5 yrs 5 ms Yes Yes Mild No No Yes
6 Female 6 yrs Yes Yes Mild-Moderate No No Yes
7 Male 4 yrs 9 ms Yes Yes Mild No No Yes
8 Female 6 yrs 1 ms Yes Yes Moderate No No Yes
9 Female 6 yrs Yes Yes Mild No No Yes
10 Male 4 yrs Yes Yes Moderate No No Yes
11 Male 5 yrs 4 ms No: 47 XYY N.D. Mild-Moderate No PDD-NOS Yes
12 Female 2 yrs 9 ms Yes N.D. Mild No No Yes
13 Male 6 yrs 4 ms Yes Yes Not testable No PDD-NOS Yes
14 Male 16 yrs 3 ms Yes Yes Moderate No No Yes
15 Male 6 yrs Yes Yes Mild No No Yes
16 Male 11 yrs Yes Yes Severe No No Yes
17 Male 6 yrs 9 ms Yes Yes Mild No No Yes
18 Male 1 yrs 9 ms Yes Yes Moderate-Mild Yes No Yes
19 Male 7 yrs Yes Yes Moderate No No Yes
20 Female 5 yrs 3 ms Yes Yes Mild No No Yes
21 Female 1 yrs 5 ms Yes Yes Moderate No No Yes
22 Male 4 yrs Yes Yes Borderline No No Yes
23 Female 8 yrs 9 ms Yes Yes Mild No No Yes
24 Male 13 yrs 7 ms Yes Yes Moderate Yes No Yes
25 Male 9 yrs 10 ms Yes Yes Moderate No PDD-NOS Yes
26 Male 10 yrs 2 ms Yes Yes Borderline No Autism Yes
27 Male 9 yrs 10 ms Yes Yes Moderate No Autism Yes
28 Male 5 yrs 9 ms Yes Yes Mild No No Yes
29 Male 4 yrs 6 ms Yes Yes Mild No No Yes
30 Male 4 yrs 7 ms Yes Yes Borderline No No Yes
31 Female 3 yrs 7 ms Yes Yes Mild No No Yes
32 Maleb 3 yrs 2 ms Yes Yes Mild No No Yes
33 Maleb 6 yrs 4 ms Yes Yes Mild No No Yes
34 Male 4 yrs 9 ms Yes Yes Moderate No No Yes
35 Female 3 yrs 6 ms Yes No; a Borderline No Autism Yes
36 Male 1 yrs 11 ms Yes Yes Moderate No No Yes
37 Female 7 yrs 6 ms Yes Yes Moderate No No Yes
38 Male 9 yrs 3 ms Yes Yes Severe Yes No Yes
39 Male 7 yrs 10 ms Yes Yes Mild No No Yes
40 Male 2 yrs 6 ms Yes Yes Moderate No No Yes
41 Male 3 yrs 10 ms Yes Yes Borderline No Autism Yes
42 Male 10 yrs Yes Yes Borderline No No Yes
43 Male 8 yrs 7 ms Yes Yes Moderate No No Yes
44 Male 5 yrs 8 ms Yes Yes Mild No No Yes
45 Male 5 yrs Yes Yes Moderate-Severe No Autism Yes
46 Male 10 yrs Yes Yes Mild No No Yes
47 Male 5 yrs 1 ms Yes Yes Moderate-Mild No No Yes
48 Female 4 yrs 9 ms Yes Yes Moderate No Autism Yes
49 Male 4 yrs 5 ms Yes Yes Profound Yes No Yes
50 Male 4 yrs 6 ms Yes Yes Mild-Moderate No No Yes
51 Female 3 yrs Yes Yes Mild No No Yes
52 Male 2 yrs 2 ms Yes Yes Borderline Yes No Yes
53 Male 13 yrs 8 ms Yes Yes Severe Yes No Yes
54 Male 12 yrs Yes Yes Mild-Moderate Yes No Yes
55 Male 3 yrs 10 ms Yes N.D. Mild No No Yes
56 Female 5 yrs 9 ms Yes Yes Mild No No Yes
57 Male 10 yrs 7 ms Yes Yes Mild No No Yes
58 Female 3 yrs 3 ms Yes Yes Moderate No Autism Yes
59 Male 17 yrs Yes Yes Profound No PDD-NOS Yes
60 Male 12 yrs 4 ms Yes Yes Moderate No No Yes
(continued on next page)

Please cite this article in press as: Battaglia A, et al., Confirmation of chromosomal microarray as a first-tier clinical diagnostic
test for individuals with developmental delay, intellectual disability, autism spectrum disorders and dysmorphic features,
European Journal of Paediatric Neurology (2013), http://dx.doi.org/10.1016/j.ejpn.2013.04.010
6 e u r o p e a n j o u r n a l o f p a e d i a t r i c n e u r o l o g y x x x ( 2 0 1 3 ) 1 e1 1

Table 3 e (continued )
Case Sex Age at Previously normal Previously Severity of Epilepsy Autism Dysmorphic
diagnosis karyotype/fish normal DD/ID features
fraxa/E

61 Female 2 yrs 2 ms No: 46, N.D. Severe Yes No Yes

XX,del(1)
(q43-qter)
62 Male 5 yrs 4 ms Yes Yes Borderline No Autism Yes
63 Male 14 yrs Yes Yes Moderate-Mild No No Yes
64 Female 2 yrs 3 ms Yes Yes Borderline-Mild Yes No Yes
65 Male 1 yr 10 ms Yes N.D. Mild No No Yes
66 Male 4 yrs 4 ms Yes Yes Borderline No PDD-NOS Yes
67 Male 4 yrs Yes Yes Moderate No PDD-NOS Yes
68 Male 8 yrs 10 ms Yes Yes Severe Yes PDD-NOS Yes
69 Male 6 yrs 1 m Yes Yes Severe No Autism Yes
70 Female 6 yrs 3 ms Yes Yes Moderate-Mild No No Yes
71 Female 11 ms N. D. N.D. Mild No No Yes
72 Female 8 yrs 5 ms Yes N.D. Moderate-Severe Yes No Yes
73 Female 4 yrs 7 ms Yes Yes Mild No No Yes
74 Male 10 yrs Yes Yes Moderate No No Yes
75 Female 4 yrs 4 ms No; N.D. Borderline No No Yes
Robertsonian 13;
14 translocation at
amniocentesis
76 Male 4 yrs 9 ms Yes Yes Borderline No PDD-NOS Yes
77 Female 11 yrs 4 ms Yes Yes Moderate Yes No Yes

Case Method and CNV Size Origin/

effective resolution (ISCN 2009)a segregation
array-CGH

1 o-array; 250 Kb arr 1p36.33(799622e2230679)x1dn, 1.4 Mb Both de novo

arr 1p36.33p36.32(2270694e3823263)x3 dn 1.5 Mb
2 o-array; 250 Kb arr 12q23.3(103031686e103581281)x1 dn, 550 Kb Both de novo
arr 12q23.3q24.12(105139121e110352856)x1 dn 5.2 Mb
3 o-array; 250 Kb arr 6p25.1(4651403e5558434)x1 mat 907 Kb Maternal segr.
4 o-array; 250 Kb arr 6p25.1(4651403e5558434)x1 mat 907 Kb Maternal segr.
5 o-array; 250 Kb arr 1p21.3(97958607e98519040)x1 dn 560 Kb de novo
6 o-array; 250 Kb arr 1p36.33(554268e2134018)x1 dn 1.58 Mb de novo
7 o-array; 250 Kb arr 18q21.31(51185159e51501491)x1 dn 316 Kb de novo
8 o-array; 250 Kb arr 14q13.1q21.1(33317408e39413341)x1 dn 6 Mb de novo
9 o-array; 250 Kb arr 2p25.2p24.3(5511851e16027633)x1 dn 10.5 Mb de novo
10 o-array, 75 Kb arr 17p11.2(16543885e20133761)x1 dn 3.5 Mb de novo
11 o-array; 250 Kb arr 17p11.2(14052497e15382791)x1 pat, 1.3 Mb Paternal segr.
arr Yp11.32q12(1264894e59023250)x3 dn All chromosome Y de novo
12 o-array, 75 Kb arr 15q13.2q13.3(28725507e30298155)x1 dn, 1.6 Mb Both de novo
arr 17p11.2(16763408e20133761)x1 dn 3.4 Mb
13 o-array, 40 Kb arr Xq27.3 (143977849e144666834)x2 mat 700 Kb Maternal segr.
14 o-array; 250 Kb arr 22q13.32q13.33(47436447e49468408)x1 dn 2 Mb de novo
15 o-array; 250 Kb arr 18p11.32p11.21(170299e13875315)x1 dn 14 Mb de novo
16 BAC-array; 1 Mb arr 9q13q21.13(70513772e75747510)x1 5 Mb N.i.
17 o-array; 250 Kb arr 5q22.1q22.2(111506045e111784628)x3 dn 278 Kb de novo
18 o-array, 75 Kb arr 4p16.34p15.33 (62447e11010859)x1 dn 11 Mb de novo
19 o-array; 250 Kb arr 8q24.22q24.3(135125129e142030490)x1 mat, 7 Mb Both maternal origin
arr 8q24.3 (142211510e142570389)x1 mat 359 Kb
20 o-array; 250 Kb arr 6q16.3q21 (104560825e109625644)x1 dn 5 Mb de novo
21 o-array; 250 Kb arr 1q24.2q31.2 (169094039e190009333)x1 21 Mb N.i.
22 o-array; 250 Kb arr 18q11.2q12.1(23237837e23819557)x3 pat 582 Kb Paternal segr.
23 o-array; 250 Kb arr 2p21(47221830e47654356)x3 pat 400 Kb Paternal segr.
24 o-array; 250 Kb arr 13q33.3q34(106808276e114077122)x1 dn 7 Mb de novo
25 o-array; 250 Kb arr 2q37.3(242639810e242757116)x1 100 Kb N.i.
26 o-array; 250 Kb arr 3q26.1q26.33(165126485e182186439)x3 dn 17 Mb de novo
27 o-array; 250 Kb arr 15q11.2(20335887e20636537)x3 pat 300 Kb Paternal segr.
28 SNP-array; 75 Kb arr 4q21.1(77973643e78325578)x3 dn 352 Kb de novo
29 o-array; 250 Kb arr 13q21.33q31.1(67762170e79276611)x1 dn 11.5 Mb de novo
30 o-array, 75 Kb arr 4q35.1q35.2(185375724e191121254)x1 dn, 6 Mb Both de novo
arr 9p24.3p22.1(1147109ee19501583) x3 dn 18 Mb

Please cite this article in press as: Battaglia A, et al., Confirmation of chromosomal microarray as a first-tier clinical diagnostic
test for individuals with developmental delay, intellectual disability, autism spectrum disorders and dysmorphic features,
European Journal of Paediatric Neurology (2013), http://dx.doi.org/10.1016/j.ejpn.2013.04.010
 e u r o p e a n j o u r n a l o f p a e d i a t r i c n e u r o l o g y x x x ( 2 0 1 3 ) 1 e1 1 7

Table 3 e (continued )
Case Method and CNV Size Origin/
effective resolution (ISCN 2009)a segregation
array-CGH

31 o-array; 250 Kb arr 1q21.1(145031367e146201635)x3 dn 1.2 Mb de novo

32 o-array; 250 Kb arr Xq22.3(104614532e105859467)x1 mat 1.2 Mb Maternal segr.
33 o-array; 250 Kb arr Xq22.3(104614532e105859467)x1 mat 1.2 Mb Maternal segr.
34 o-array; 250 Kb arr Yq11.221q11.223(15924921e23283748)x2 pat 7.4 Mb Paternal segr.
35 o-array; 250 Kb arr 16p13.11(14956252e16157108)x3 pat 1.2 Mb Paternal segr.
36 o-array; 250 Kb arr 15q13q13.3(28725507e30701432)x3 mat 2 Mb Maternal segr.
37 o-array; 250 Kb arr 16p11.2p12.2(21382561e29581514)x1 dn 8.2 Mb de novo
38 o-array; 250 Kb arr 17q25.3(70429779e78623230)x3 mat, 8 Mb t(17; 21) (q25.3; q22.3)mat
arr 21q22.3(40161399e46892352)x1 mat 7 Mb
39 SNP-array; 75 Kb arr 4q35.2(187568520e187767160)x3 pat, 199 Kb Both paternal segr.
arr 20q12(40696758e40789088)x1 pat 92 Kb
40 o-array; 250 Kb arr 17q12(26048537e27391327)x1 dn 1.3 Mb de novo
41 o-array, 75 Kb arr 22q11.21( 17276972e19712953)x3 3 Mb N.i.
42 o-array; 250 Kb arr 14q22.1(49645321e50022606)x3 mat 377 Kb Maternal origin
43 o-array; 250 Kb arr 16p13.11(14852061e16157108)x1 pat, 1.3 Mb Paternal segr.
arr 19p13.3(5106515e5592847)x3 mat 486 Kb Maternal segr.
44 o-array; 250 Kb arr 17q25.1(70183432e71157411)x3 mat 1 Mb Maternal origin
45 o-array; 250 Kb arr Xp22.31(6561155e7187444)x2 mat 626 Kb Maternal origin
46 o-array; 250 Kb arr 1q43q44(240318783e242286969)x1 dn 2 Mb de novo
47 o-array; 250 Kb arr 11p14.1p12(27730989e38824714)x1 dn 11 Mb de novo
48 o-array; 250 Kb arr 16p12.2(21507188e21745052)x3 mat 238 Kb Maternal segr.
49 o-array, 75 Kb arr 10p12.31p11.22(22679698e32377501)x1 no mat 10 Mb Mother excl
50 o-array; 250 Kb arr 12p13.33p13.32(2672274e3851960)x1 dn 1.2 Mb de novo
51 o-array; 250 Kb arr 17p11.2(16543855e20133761)x3 dn 3.6 Mb de novo
52 o-array; 250 Kb arr 1p36.33p36.32(554327e4922668)x1 4.4 Mb N.i.
53 o-array; 250 Kb arr 1p36.33p36.22(1239023e10655356)x1 no mat 10 Mb Mother excl
54 o-array; 250 Kb arr 16p13.21p13.13(9670361e10933583)x1 pat 1.3 Mb Paternal segr.
55 o-array, 75 Kb arr 17p13.3(1182533e2286340)x1 dn 1.1 Mb de novo
56 o-array; 250 Kb arr 7q36.1(151204610e152087619)x3 1 Mb N.i.
57 o-array, 75 Kb arr 7q35(146129853e146361435)x1 pat 231 Kb Paternal origin
58 o-array; 250 Kb arr 6q24.3(145883327e146734394)x3 mat 291 Kb Maternal origin
59 o-array; 250 Kb arr 3p21.31p21.1(48015103e53190621)x3 5.1 Mb N.i.
60 o-array, 75 Kb arr 4q35.2(189484696e189745394)x1 pat, 261 Kb Both paternal segr.
arr 7q35(146106661e146426065)x1 pat 219 Kb
61 o-array, 75 Kb arr 1q43q44(235323717e247195039)x1 dn, 11.9 Mb Both de novo
arr 5p15.33(75178e874908)x3 dn 800 Kb
62 o-array, 75 Kb arr 15q13.1q13.2(26999772e28370728)x1 pat 1.37 Mb Paternal segr.
63 o-array, 75 Kb arr 18q12.1(27728909e27847543)x3 mat 119 Kb Maternal segr.
64 SNP-array; 75 Kb arr 8q24.3(142290961e143012170)x3 mat 721 Kb Maternal segr.
65 SNP-array; 75 Kb arr 18q12.1(26036013e26180676)x3 mat 144 Kb Maternal segr.
66 SNP-array; 75 Kb arr 11p15.2(14646299e14819024)x1 pat 172 Kb Paternal segr.
67 SNP-array; 75 Kb arr 12p12.1p12.2(20898999e21301357)x1 mat, 402 Kb Maternal segr.
arr 22q13.33(48960804e49581310)x1 dn 615 Kb de novo
68 o-array; 250 Kb arr 2q13(110811831e112908339)x1 dn, 2.1 Mb de novo
arr 20q12(40513019e40686494)x1 mat 174 Kb Maternal segr.
69 SNP-array; 75 Kb arr 5q35.1(171422562e171553820)x1 pat 131 Kb Paternal segr.
70 SNP-array; 75 Kb arr 9p24.3p23(27747e12410023)x1 dn 12.4 Mb de novo
71 SNP-array; 75 Kb arr 6q21q22.31(112781909e121626879)x1 w 2 dn, 8.9 Mb Both de novo
arr 6q21(105899127e107535302)x1 dn 1.6 Mb
72 SNP-array; 75 Kb arr 6q25.2q27(153930435e166455746)x1 dn 12.7 Mb de novo
73 SNP-array; 75 Kb arr 16q21(56260206e56362678)x3 pat 102 Kb Paternal segr.
74 SNP-array; 75 Kb arr 1p34.3(35224053e36420094)x1 dn 1.2 Mb de novo
75 SNP-array; 100 Kb arr Xp22.31(6046480e6465519)x3 mat, 419 Kb Both maternal segr.
arr Xq28(147911730e148431350)x3 mat 519 Kb
76 SNP-array; 75 Kb arr 22q11.21(17256416e17404795)x1 mat 148 Kb Maternal segr.
77 SNP-array; 75 Kb arr Xq28(153225755e153357171)x3 dn 131 Kb de novo

Case Genes OMIM

morbid

1 56 RefSeq AGRN; GABRD; SKY

31 RefSeq PEX10
(continued on next page)

Please cite this article in press as: Battaglia A, et al., Confirmation of chromosomal microarray as a first-tier clinical diagnostic
test for individuals with developmental delay, intellectual disability, autism spectrum disorders and dysmorphic features,
European Journal of Paediatric Neurology (2013), http://dx.doi.org/10.1016/j.ejpn.2013.04.010
8 e u r o p e a n j o u r n a l o f p a e d i a t r i c n e u r o l o g y x x x ( 2 0 1 3 ) 1 e1 1

Table 3 e (continued )
Case Genes OMIM
morbid

2 NFYB; TXNRD1; RPL18AP3; CHST11 No

57 RefSeq ISCU; DAO; MMAB; MVK; TRPV4; ATP2A2; MYL2;SART3
3 CDYL; RPP40; PPP1R3G; LYRM4; FARS2 No
4 CDYL; RPP40; PPP1R3G; LYRM4; FARS2 No
5 DPYD; MIR137 DPYD
6 59 RefSeq AGRN; GABRD
7 TCF4 TCF4
8 38 RefSeq CFL2; PAX9; MIPOL1; SEC23A; SIP1; FBXO33
9 41 RefSeq KLF11; LPIN1;MYCN; ODC1
10 64 RefSeq FLCN; RAI1; ATPAF2; MYO15A; ALDH3A2; AKAP10
11 8 RefSeq PMP22
360 RefSeq USP9Y
12 8 RefSeq TRPM1; CHRNA7;
64 RefSeq FLCN; RAI1; ATPAF2; MYO15A; ALDH3A2; AKAP10
13 SPANXN1 No
14 35 RefSeq ALG12; MLC1; SCO2; ARSA; SHANK3
15 62 RefSeq LPIN2; NDUFV2; AFG3L2; MC2R
16 25 RefSeq FXN; TJP2; TMC1
17 EPB41L4A; NCRNA00219; SNORA13 No
18 114 RefSeq 16 OMIM Morbid
19 14 ReFSeq KCNK9; TRAPPC9
DENND3; SLC45A4; GPR20; PTP4A3; SNORD5 No
20 29 RefSeq AIM1; PDSS2; SOBP; SEC63; OSTM1
21 121 RefSeq 13 OMIM Morbid
22 CHD2 No
23 7 RefSeq EPCAM; MSH2
24 39 RefSeq IRS2; COL4A1; ING1; GRK1
25 CICP10 No
26 71 RefSeq DNAJC19; GHSR; PDCD10; PIK3CA; SERPIN1; SI; SPATA16
27 TUBGCP5; CYFIP1; NIPA2; NIPA1 NIPA1
28 ANKRD6; SEPT11; CCNI; CCNG2 No
29 32 RefSeq CLN5; EDNRB
30 29 RefSeq SLC25A4; CYP4V2; KLKB1; F11
66 RefSeq VLDLR; KCNV2; GLIS3; JAK2; GLDC; TYRP1; FREM1
31 15 RefSeq GJA5; GJA8
32 CXorf57; IL1RAPL2; MUM1L1; NRK; RNF128;SERPINA7 No
33 CXorf57; IL1RAPL2; MUM1L1; NRK; RNF128;SERPINA7 No
c c
34
35 15 RefSeq MYH11
36 10 RefSeq TRPM1; CHRNA7
37 71 RefSeq ATP2A1; CLN3; COG7; IL21R; IL4R; OTOA, PALB2; SCNN1B; SCNN1G; TUMF
170 RefSeq 19 OMIM Morbid
38 109 RefSeq CBS; COL18A1; COL6A1; COL6A2; CSTB; FTCD; FTCD; PCNT; TMPRSS3
39 MTNR1A; FAT1 No
PTPRT No
40 17 RefSeq NF1
41 56 RefSeq COMT; GP1BB; PRODH; RTN4R; SERPIND1; SNAP29; TBX1
42 C14orf138; SOS2; L2HGDH; ATPS5; CDKL1; MAP4K5 L2HGDH
43 17 RefSeq MYH11; ABCC6
PLCA2; PTPR5; No
SAFB; SAFB2; ZNRF4
44 33 RefSeq SLC25A19; SLC9A3R1; TSEN54; USH1G
45 HDHD1; STS STS
46 PLD5; RSL24D1P4; No
CEP170; SDCCAG8;
AKT3; ZNF238
47 48 RefSeq FSHB; PAX6; WT1
48 IGSF6; OTOA
METTL9; OTOA
49 50 RefSeq PTF1A; MYO3A; PDSS1; MTPAP;
MAP3K8; ZEB1
50 13 RefSeq CACNA1C
51 64 RefSeq FLCN; RAI1; ATPAF2; MYO15A;
ALDH3A2; AKAP10

Please cite this article in press as: Battaglia A, et al., Confirmation of chromosomal microarray as a first-tier clinical diagnostic
test for individuals with developmental delay, intellectual disability, autism spectrum disorders and dysmorphic features,
European Journal of Paediatric Neurology (2013), http://dx.doi.org/10.1016/j.ejpn.2013.04.010
 e u r o p e a n j o u r n a l o f p a e d i a t r i c n e u r o l o g y x x x ( 2 0 1 3 ) 1 e1 1 9

Table 3 e (continued )
Case Genes OMIM
morbid

52 85 RefSeq AGRN; GABRD; PEX10

53 114 RefSeq GABRD; PEX10; NPHP4; ESPN; PLEKHG5;
PARK7; H6PD; KIF1B; PEX14
54 7 RefSeq No
55 31 RefSeq R1LP; PRPF8; SERPINF2; PAFAH1B1
56 11 RefSeq PRKAG2
57 CNTNAP2 CNTNAP2
58 EPM2A; FBXO30; SHPRH; EPM2A
GRM1; FUNDC2P3
59 148 RefSeq 15 OMIM Morbid
60 LOC401164 No
CNTNAP2 CNTNAP2
61 104 RefSeq FH; NLRP3; RYR2
41 RefSeq MTRR; NDUFS6; SDHA;
SLC6A19; SLC6A3; TERT
62 APBA2; FAM189A1; NDNL1; No
TJP1; GOLGA8J; FAM7A3
63 TRAPPC8 No
64 SLC45A4; GPR20; No
PTP4A3; SNORD5
65 MIR302F No
66 PDE3B No
67 SLCO1B1; SLCO1B3; No
SLCO1B7
28 RefSeq SCO2; ARSA; SHANK3
68 RGPD6; BUB1; ACOXL; MERTK
BCL2L11; ANAPC1;
MERTK; TMEM87B; FBLN7;
ZC3H6; ZC3H8; RGDP8
PTPRT No
69 STK10 No
70 42 RefSeq DOCK8; KANK1; VLDLR;
KCNV2; GLIS3; JAK2; GLDC
71 33 RefSeq TSPYL1; RSPH4A; PLN
PREP; PRDM1; AIM1; AIM1
RTN4IP1; QRSL1;
C6orf203; BEND3
72 45 RefSeq GTF2H5; SOD2; PLG; PARK2
73 GPR97; CCDC135; No
KATNB1; KIFC3
74 19 RefSeq COL8A2
75 NLGN4X; VCX3A No
IDS;CXorf40A No
76 DGCR6; PRODH; DCGR2 PRODH
77 10 RefSeq FLNA; EMD; TAZ; GDI1

Legend yrs: years; ms: months; segr.: segregation; N.i.: no information available; N.D.: not determined; o-array: oligonucleotide-array.
a Based on Release hg18.
b Patients n. 3 and 4 are dyzigotic twins; patients n. 32 and 33 are brothers; a: premutation allele at the FRAXA locus (61-62 triplets).
c Chromosome Y genes were not considered as pathogenic in our study.

among those with borderline/mild/moderate ID6 we observed
a higher yield in the latter group (65/77 versus 12/77).
Our study results are inherently limited by the different
CMA platforms used and the varying knowledge of the
parental phenotype. There are, however, several strengths
represented by the comprehensive phenotypic character-
ization by a single pair of clinicians (A.B. and J.C.C.) at a
single tertiary center (Stella Maris Clinical Research Insti-
tute), the prior exhaustive evaluation including high reso-
lution banding, molecular analysis at the FRAXA/E loci,
and the standardized psychopathological assessment for
DD/ID/ASDs.
Our study provides further evidence of the high diagnostic
yield of CMA for genetic testing of individuals with unex-
plained DD/ID/ASDs and dysmorphic features, confirming its
first-tier usefulness in such conditions. This technology ap-
pears to be capable of finding the cause of DD/ID/ASDs in a
much greater number of affected individuals (w23%) as is
conventional cytogenetic analysis (w3%, excluding Down
syndrome and other recognizable chromosomal syndromes).17

Please cite this article in press as: Battaglia A, et al., Confirmation of chromosomal microarray as a first-tier clinical diagnostic
test for individuals with developmental delay, intellectual disability, autism spectrum disorders and dysmorphic features,
European Journal of Paediatric Neurology (2013), http://dx.doi.org/10.1016/j.ejpn.2013.04.010
10 e u r o p e a n j o u r n a l o f p a e d i a t r i c n e u r o l o g y x x x ( 2 0 1 3 ) 1 e1 1
Table 4 e Percentage for the different CNVs categories found in the entire sample, in the borderline ID, in the mixed ID, and
in the DD/ID/ASDs patients.
Entire sample Borderline ID
Causative CNVs 65/91 (71.4%) 3/7
Probably causative CNVs 8/91 (8.8%) 2/7
Uncertain CNVs 5/91 (5.5%) 0/7
Non causative CNVs 13/91 (14.3%) 2/7
High resolution screening using microarray not only detects
submicroscopic chromosomal imbalances, but also allows ac-
curate delineation of the duplicated or deleted chromosomal
segment. This is crucial for phenotypeegenotype correlation
and for identifying candidate genes involved in the develop-
ment of certain anomalies and of ID/ASDs.
The additional CNVs detected in 5/77 (6.49%) patients with
an already known chromosomal disorder, support the rele-
vance of the clinical observation even in the era of CMA.
We were able to analyze only a small group of patients with
SNP-array platform, due to its very recent introduction as a
clinical tool. Anyway, the opportunity to evaluate the rate of
homozigosity throughout the genome appears promising to
unmask recessive form of ID/ASDs and identify new AR dis-
ease-genes.
5. Conclusions
Our experience shows once more that a high percentage of
DD/ID/ASDs/dysmorphic features cases hitherto considered
idiopathic is caused by submicroscopic chromosomal im-
balances. This, together with the literature data,17 and the
known cost-effectiveness,34 strongly supports the use of CMA
in place of standard karyotyping as the first-tier cytogenetic
diagnostic test in the clinical evaluation of children with DD/
ID/ASDs and dysmorphic features.
Funding source
This study was supported by grants from the Italian Ministry
of Health (to LB and AB).
Financial disclosure
The authors have no financial relationships relevant to this
article to disclose.
Contributors statement
Agatino Battaglia MD: Dr. Battaglia conceptualized and
designed the study, drafted the initial manuscript, reviewed
and revised the manuscript, and approved the final manu-
script as submitted.
Viola Doccini MD: Dr. Doccini carried out the initial anal-
ysis, revised the manuscript, and approved the final manu-
script as submitted.
Please cite this article in press as: Battaglia A, et al., Confirmation of chromosomal microarray as a first-tier clinical diagnostic
test for individuals with developmental delay, intellectual disability, autism spectrum disorders and dysmorphic features,
European Journal of Paediatric Neurology (2013), http://dx.doi.org/10.1016/j.ejpn.2013.04.010
Mixed ID ID/ASDs patients
(42.8%) 51/64 (79.69%) 11/20 (55%)
(28.6%) 2/64 (3.11%) 4/20 (20%)
(0.0%) 3/64 (4.7%) 2/20 (10%)
(28.6%) 8/64 (12.5%) 3/20 (15%)
Laura Bernardini PhD: Dr. Bernardini carried out the CMA,
confirmed its results, revised the manuscript, and approved
the final manuscript as submitted.
Antonio Novelli PhD: Dr. Novelli coordinated and super-
vised data collection at the Mendel institute, revised the
manuscript, and approved the final manuscript as submitted.
Sara Loddo PhD: Dr. Loddo carried out the CMA, confirmed
its results, revised the manuscript, and approved the final
manuscript as submitted.
Anna Capalbo PhD: Dr. Capalbo carried out the CMA,
confirmed its results, revised the manuscript, and approved
the final manuscript as submitted.
Tiziana Filippi MD: Dr. Filippi carried out the initial anal-
ysis, revised the manuscript, and approved the final manu-
script as submitted.
John C. Carey MD: Dr. Carey contributed to design the
study, critically reviewed the manuscript, and approved the
final manuscript as submitted.
Conflict of interest
The authors have no conflict of interest to disclose.
Acknowledgment
This study was supported by grants from the Italian Ministry
of Health to AB and LB.
references
1. Shevell MI, Ashwal S, Donley D, et al. Practice parameter:
evaluation of the child with global developmental delay:
report of the quality standards subcommittee of the
American academy of neurology and the practice
committee of the child neurology society. Neurology
2003;60:367e80.
2. Stankiewicz P, Beaudet AL. Use of array CGH in the evaluation
of dysmorphology, malformations, developmental delay, and
idiopathic mental retardation. Curr Opin Genet Dev
2007;17:182e92.
3. Luckasson R, Borthwick-Duffy S, Buntinx WHE, et al. Mental
retardation: definition, classification, and systems of supports. 10th
ed. Washington, DC: American Association on Mental
Retardation (BEST); 2002.
4. Moeschler JB. Medical genetics diagnostic evaluation of the
child with developmental delay or intellectual disability. Curr
Opin Neurol 2008;21:117e22.
 e u r o p e a n j o u r n a l o f p a e d i a t r i c n e u r o l o g y x x x ( 2 0 1 3 ) 1 e1 1
5. CDC: centers for disease control and prevention. About intellectual
disability, from: http://www.cdc.gov/ncbddd/dd/ddmr.htm;
2005.
6. Battaglia A, Carey JC. Diagnostic evaluation of developmental
delay/mental retardation: an overview. Am J Med Genet (Semin
Med Genet) 2003;117C:3e14.
7. National Institute of Mental Health. Autism spectrum
disorders (pervasive developmental disorders). Available at:
http://www.nimh.nih.gov/health/publications/autism/
nimhautismspectrum.pdf. [accessed 13.07.10].
8. Economic CDC. Costs associated with mental retardation,
cerebral palsy, hearing loss, and vision impairment e United
States, 2003. MMWR Morb Mortal Wkly Rep 2004;53:57e9.
9. McLaren J, Bryson SE. Review of recent epidemiological
studies of mental retardation: prevalence, associated
disorders, and etiology. Am J Ment Retard 1987;92:243e54.
10. Battaglia A, Bianchini E, Carey JC. Diagnostic yield of the
comprehensive assessment of developmental delay/mental
retardation in an institute of child neuropsychiatry. Am J Med
Genet 1991;82:60e6.
11. Schaefer GB, Bodensteiner JB. Evaluation of the child with
idiopathic mental retardation. Pediatr Clin North Am
1992;39:929e43.
12. First LR, Palfrey JS. The infant or young child with
developmental delay. N Engl J Med 1994;330:478e83.
13. Majnemer A, Shevell MI. The diagnostic yield of the
neurologic assessment of the developmentally delayed child.
J Pediatr 1995;127:193e9.
14. Flint J, Wilkie AO. The genetics of mental retardation. Br Med
Bull 1996;52:453e64.
15. Shevell MI, Majnemer A, Rosenbaum P, Abrahamowicz M.
Etiologic yield of subspecialists evaluation of young children
with global developmental delay. J Pediatr 2000;136:593e8.
16. Srour M, Mazer B, Shevell MI. Analysis of clinical features
predicting etiologic yield in the assessment of global
developmental delay. Pediatrics 2006;118:139e45.
17. Miller DT, Adam MP, Aradhya S, et al. Consensus statement:
chromosomal microarray is a first-tier clinical diagnostic test
for individuals with developmental disabilities or congenital
anomalies. Am J Hum Genet 2010;86:749e64.
18. Solinas-Toldo S, Lampel S, Stilgenbauer S, et al. Matrix-based
comparative genomic hybridization: biochips to screen for
genomic imbalances. Genes Chromosomes Cancer
1997;20:399e407.
19. Lord C, Rutter M, LeCouteur A. Autism diagnostic review-
revised: a revised version of a diagnostic interview for
caregivers of individuals with possible pervasive
developmental disorders. J Autism Dev Disord 1994;24:659e85.
20. Lord C, Rutter M, DiLavore PC, Risi S. Autism diagnostic
observation schedule. Los Angeles: Western Psychological
Services; 2002.
21. Bernardini L, Castori M, Capalbo A, et al. Syndromic
craniosynostosis due to complex chromosome 5
Please cite this article in press as: Battaglia A, et al., Confirmation of chromosomal microarray as a first-tier clinical diagnostic
test for individuals with developmental delay, intellectual disability, autism spectrum disorders and dysmorphic features,
European Journal of Paediatric Neurology (2013), http://dx.doi.org/10.1016/j.ejpn.2013.04.010
11
rearrangement and MSX2 gene triplication. Am J Med Genet
2007;143A:2937e43.
22. Carbone A, Bernardini L, Valenzano F, et al. Array-based
comparative genomic hybridization in early-stage mycosis
fungoides: recurrent deletion of tumor suppressor genes
BCL7A, SMAC/DIABLO, and RHOF. Genes Chromosomes Cancer
2008;47:1067e75.
23. Kearney HM, Thorland EC, Brown KK, Quintero-Rivera F,
South ST., Working Group of the American College of Medical
Genetics Laboratory Quality Assurance Committee. American
college of medical genetics standards and guidelines for
interpretation and reporting of postnatal constitutional copy
number variants. Genet Med 2011 Jul;13:680e5.
24. Kearney HM, Kearney JB, Conlin LK. Diagnostic implications
of excessive homozygosity detected by SNP-based
microarrays: consanguinity, uniparental disomy, and
recessive single-gene mutations. Clin Lab Med
2011;31:595e613.
25. Sund KL, Zimmerman SL, Thomas C, et al. Regions of
homozygosity identified by SNP microarray analysis aid in
the diagnosis of autosomal recessive disease and incidentally
detect parental blood relationships. Genet Med 2013;15:70e8.
26. Mikhail FM, Lose EJ, Robin NH, et al. Clinically relevant single
gene or intragenic deletions encompassing critical
neurodevelopmental genes in patients with developmental
delay, mental retardation, and/or autism spectrum disorders.
Am J Med Genet 2011;155:2386e96.
27. Bowtell D, Fu P, Simon M, Senior P. Identification of murine
homologues of the Drosophila son of sevenless gene:
potential activators of ras. Proc Natl Acad Sci U S A 1992 Jul
15;89:6511e5.
28. Liu AM, Lo RK, Lee MM, et al. Galpha16 activates ras by
forming a complex with tetratricopeptide repeat 1 (TPR1) and
Son of Sevenless (SOS). Cell Signal 2010;22:1448e58.
29. Miles JH. Autism spectrum disorders- a genetics review. Genet
Med 2011;13:278e94.
30. Valnegri P, Montrasio C, Brambilla D, et al. The X-linked
intellectual disability protein IL1RAPL1 regulates excitatory
synapse formation by binding PTPd and RhoGAP2. Hum Mol
Genet 2011;20:4797e809.
31. Menten B, Maas N, Thienpont B, et al. Emerging patterns of
cryptic chromosomal imbalances in patients with idiopathic
mental retardation and multiple congenital anomalies: a new
series of 140 patients and review of the literature. J Med Genet
2006;43:625e33.
32. Van Ommen GJ. Frequency of copy number variation in
humans. Nat Genet 2005;37:333e4.
33. Girirajan S, Rosenfeld JA, Cooper GM, et al. A recurrent
16p12.1 microdeletion supports a two-hit model for severe
developmental delay. Nat Genet 2010;42.3:203e9.
34. Trakadis Y, Shevell M. Microarray as a first genetic test in
global developmental delay: a cost-effectiveness analysis. Dev
Med Child Neurol 2011;53:994e9.